DN Cells Research Articles

Delta-like 4-mediated Notch signaling is required for early T-cell development in a three-dimensional thymic structure.

Delta-like 4 (Dll4)-mediated Notch signaling is critical for specifying T-cell fate, but how Dll4-mediated Notch signaling actually contributes to T-cell development in the thymus remains unclear. To explore this mechanism in the thymic three-dimensional structure, we performed fetal thymus organ culture using Dll4-deficient mice. DN1a/b+DN2mt cells, which had not yet committed to either the αβ T or γδ T/NK cell lineage, did not differentiate into the αβ T-cell lineage in Dll4-deficient thymus despite the lack of cell fate conversion into other lineages. However, DN3 cells efficiently differentiated into a later developmental stage of αβ T cells, the double-positive (DP) stage, although the proliferation was significantly impaired during the differentiation process. These findings suggest that the requirement for Notch signaling differs between the earliest and pre-TCR-bearing precursors and that continued Notch signaling is required for proper differentiation with active proliferation of αβ T lineage cells. Furthermore, we showed that Notch signaling increased the c-Myc expression in DN3 cells in the thymus and that its overexpression rescued the proliferation and differentiation of DN3 cells in the Dll4-null thymus. Therefore, c-Myc plays a central role in the transition from stage DN3 to DP as a downstream target of Notch signaling.

Bim expression in early DP thymocytes limits development of TCRαβ+DN thymocytes and CD8αα intestinal intraepithelial lymphocytes (HEM2P.243)

Abstract Thymocyte negative selection is thought to cull T cells with strong self-reactivity and maintain T cells with non-self-specificity. While initial work implicated Bim as a critical mediator of thymocyte negative selection, its role in the process has been controversial. Thus, we re-examined thymocyte development in mice with global and T cell specific loss of Bim. We found that global Bim-/- mice had a striking accumulation of DN4 thymocytes expressing high levels of surface TCR, which failed to generate DP cells in OP9-DL1 culture. Interestingly, CD4Cre-driven deletion of Bim promoted DN4 cells, but later, dLckCre-driven deletion of Bim did not. Combined, these data show that the aberrant DN4 cells in Bim-/- mice were once DP cells, but had lost co-receptor expression. Investigating the fate of these cells, we found that splenic DN T and gut CD8αα T cells were increased in Bim-/- mice. While IL-15 is critical for both DN/CD8αα T cells, the additional loss of Bim partially restored DN/CD8αα T cells in IL-15 deficient mice, suggesting that IL-15 signals antagonize Bim to promote DN T and CD8αα T cells. Finally, TCR or IL-15 signals were sufficient to promote expression of both CD8αα and the gut homing receptor CCR9 on splenic DN T cells. In conclusion, these data further delineate the role of Bim on thymocyte fate and DN/CD8αα T cell development; and show that dLckCre-Bimf/f mice can isolate the effects of Bim on peripheral T cell survival without affecting thymic selection.

291. A Novel Safety Assay for Retroviral Vectors That Reproduces Lmo2 Proto-Oncogene Insertional Activation Events

Gene therapies for X-linked severe combined immunodeficiency (SCID-X1) and Wiskott-Aldrich syndrome (WAS) with gammaretroviral vectors have caused a number of cases of acute lymphoid leukemias due to insertional activation of oncogenes, mostly LMO2. Transduction of murine bone marrow hematopoietic cells and subsequent culture for myeloid immortalization or transplant into recipient mice detected the oncogenic activity of the spleen focus-forming virus (SFFV) vector, which was used in the WAS trial, but failed to detect the oncogenic activity of the SCID-X1 gene therapy vector MFG-gc. The lack of the most relevant Lmo2 insertion by gammaretroviral vectors in these assays demands more relevant and sensitive vector safety assays. It has been shown that murine early thymic progenitor cells (ETP), when cultured on OP9-DL1 stroma cells, progress through the DN1, DN2, DN3, DN4, DP and SP differentiation stages, and that overexpression of LMO2 in ETP cells caused a differentiation block at the DN2 stage (CD4-CD8-CD25+CD44+). We tested whether transduction of ETP cells with gammaretroviral or lentiviral vectors could reproduce Lmo2 integrations and induce DN2 differentiation block. We tested three different gamma-retroviral vectors MSCV-GFP, SFFV-GFP and MFG-gc and two lentiviral vectors Cl20-SFFV-mCherry and Cl20i4r-SFFV-mCherry. The two self-inactivating lentiviral vectors contain a single internal SFFV long terminal repeat but differ in that one contains the cHS400 chromatin insulators. We transduced ETP cells with these vectors and cultured them on OP9-DL1 cells for up to 40 days. The range of vector copy number was 1.07-10.78 for the MSCV-GFP vector, 0.24-1.22 for the SFFV-GFP vector, 0.59-7.98 for the MFG-gc vector, 3.97-13.04 for the Cl20-SFFV-mCherry vector and 4.57-18.21 for the Cl20i4r-SFFV-mCherry vector at days 10-11. Between days 20-35, a distinct DN2-blocked cell subpopulation was clearly detected by flow cytometry in all the 20 gamma-retroviral groups and also in 3/9 lentiviral groups. None of the 6 mock groups had any evidence of DN2 blockade. Insertion site mapping of sorted DN2 cells showed that in the 10 MSCV-GFP groups, 3 had Lmo2 insertions, 3 had Mef2c insertions, and 3 had both Lmo2 and Mef2c insertions; in the 6 SFFV-GFP groups, 3 had Lmo2 insertions; in the 4 MFG-gc groups, 3 had Lmo2 insertions; both Cl20-SFFV-mCherry groups had Mef2c insertions and the one Cl20i4r-SFFV-mCherry group had Lmo2 insertion. These insertions occurred either in the introns or within +/- 50kb window of the gene. Transplant of the DN2-blocked cells from selected MSCV-GFP groups into recipient mice led to acute lymphoid leukemias. These results show that the transduction of ETP cells and the resultant DN2 blockade represent a more relevant and sensitive assay for vector safety assessment. Our result also suggests that an ETP-like subpopulation may be present in the bone marrow of SCID-X1 and WAS patients and explain the enhanced propensity of these disorders to oncogenic transformations.

Abstract P2-04-05: Modeling luminal breast cancer heterogeneity: Combination therapies to suppress hormone receptor-negative subpopulations among receptor-positive ones

Abstract All Luminal breast cancers are heterogeneous, containing substantial numbers of estrogen (ER) and progesterone (PR) receptor-negative cells among the ER+PR+ ones. We have identified two such Luminal-derived ER–PR– cells: The first, we call "Luminobasal" (LB), are ER–PR– and cytokeratin 5 (CK5)-positive. The second, we call "Double Negative" (DN), are ER–PR– and CK5–. Currently, neither is targeted for treatment. Luminobasal cells: To address the relationships between true Luminal ER+PR+CK5– and Luminobasal ER–PR–CK5+ cells in Luminal cancers and tightly control their ratios we generated isogenic pure Luminal (pLUM) and pure Luminobasal (pLB) cells from solid tumor xenografts starting from the same parental T47Dco Luminal human breast cancer cell line. Cells were gene profiled and unique immunohistochemical (IHC) biomarkers for each were confirmed. Interaction dynamics studies show that in mixed-cell 3D colonies, pLUM or Luminal MCF-7 cells suppress growth of pLB cells. Similarly, in mixed-cell solid tumor xenografts, pLUM cells suppress pLB proliferation. Alarmingly, in mixed-cell models, monotherapy of a pLUM or MCF-7 subpopulation with the antiestrogen Fulvestrant inadvertently expands the number of pLB cells. This suggested that pLB cells also need to be treated. An 89 drug FDA-approved oncology library and high throughput screening methods were therefore used to show that pLB cells are specifically targeted by the EGFR inhibitors Gefitinib and Erlotinib. We then showed, in both mixed-cell 3D colonies and mixed-cell solid mouse tumors, that combination therapy using Fulvestrant plus Gefitinib constitutes a robust treatment strategy that targets both cell populations simultaneously. Double Negative cells: DN cells are EGFR– and therefore unaffected by EGFRi. However they express the Luminal marker Claudin-3 (CLD3). Among other conditions, DN cells arise as a discrete CLD3+ subpopulation within CLD3– pLB xenografts. This allowed us to use CLD3 for FACS isolation and purification of DN cells from xenografts. Purified cells were gene expression profiled, which showed that DN cells have a unique genomic signature that distinguishes them from LUM and LB cells. They express diagnostic markers suitable for IHC and FACS-sorting, some of which also present potential therapeutic targets. We propose that heterogeneous Luminal breast cancers may be best treated with combination therapies that include endocrine, EGFRi and/or DN inhibitors. Importantly, optimizing combination regimens that eradicate or suppress not only ER+PR+ cells but also diverse ER–PR– cells in Luminal disease requires that primary tumors be pre-screened for appropriate biomarkers, including but not limited to ER, PR, CK5 and EGFR. Citation Format: Allison L Scaling, Aaron J Knox, Maurcio P Pinto, Brian S Bliesner, James M Haughian, Hany A Abdel-Hafiz, Kathryn B Horwitz. Modeling luminal breast cancer heterogeneity: Combination therapies to suppress hormone receptor-negative subpopulations among receptor-positive ones [abstract]. In: Proceedings of the Thirty-Seventh Annual CTRC-AACR San Antonio Breast Cancer Symposium: 2014 Dec 9-13; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2015;75(9 Suppl):Abstract nr P2-04-05.

IL-7 coordinates proliferation, differentiation and Tcra recombination during thymocyte β-selection.

pre-T cell receptor (TCR) and Notch signaling induce transient self-renewal or “β-selection” of TCRβ+ CD4 CD8 double-negative-3 (DN3) and DN4 progenitors that differentiate into CD4 CD8 double positive (DP) thymocytes which then rearrange Tcra. Interleukin-7 (IL-7) promotes Bcl2-dependent survival of TCRβ− DN thymocytes, but IL-7 functions during β-selection remain unclear. Here, we show that IL-7 signals TCRβ+ DN3 and DN4 thymocytes to upregulate genes involved in cell growth and represses Bcl6. Accordingly, IL-7-deficient DN4 cells lacked trophic receptors and did not proliferate, but rearranged Tcra prematurely and differentiated rapidly. Bcl6 deletion, but not BCL2 over-expression, partially restored DN4 self-renewal in the absence of IL-7. Thus, IL-7 critically collaborates with pre-TCR and Notch signaling to coordinate proliferation, differentiation and Tcra recombination during β-selection.

SCL, LMO1 and Notch1 reprogram thymocytes into self-renewing cells.

The molecular determinants that render specific populations of normal cells susceptible to oncogenic reprogramming into self-renewing cancer stem cells are poorly understood. Here, we exploit T-cell acute lymphoblastic leukemia (T-ALL) as a model to define the critical initiating events in this disease. First, thymocytes that are reprogrammed by the SCL and LMO1 oncogenic transcription factors into self-renewing pre-leukemic stem cells (pre-LSCs) remain non-malignant, as evidenced by their capacities to generate functional T cells. Second, we provide strong genetic evidence that SCL directly interacts with LMO1 to activate the transcription of a self-renewal program coordinated by LYL1. Moreover, LYL1 can substitute for SCL to reprogram thymocytes in concert with LMO1. In contrast, inhibition of E2A was not sufficient to substitute for SCL, indicating that thymocyte reprogramming requires transcription activation by SCL-LMO1. Third, only a specific subset of normal thymic cells, known as DN3 thymocytes, is susceptible to reprogramming. This is because physiological NOTCH1 signals are highest in DN3 cells compared to other thymocyte subsets. Consistent with this, overexpression of a ligand-independent hyperactive NOTCH1 allele in all immature thymocytes is sufficient to sensitize them to SCL-LMO1, thereby increasing the pool of self-renewing cells. Surprisingly, hyperactive NOTCH1 cannot reprogram thymocytes on its own, despite the fact that NOTCH1 is activated by gain of function mutations in more than 55% of T-ALL cases. Rather, elevating NOTCH1 triggers a parallel pathway involving Hes1 and Myc that dramatically enhances the activity of SCL-LMO1 We conclude that the acquisition of self-renewal and the genesis of pre-LSCs from thymocytes with a finite lifespan represent a critical first event in T-ALL. Finally, LYL1 and LMO1 or LMO2 are co-expressed in most human T-ALL samples, except the cortical T subtype. We therefore anticipate that the self-renewal network described here may be relevant to a majority of human T-ALL.

PRL2 Phosphatase Regulate Early Stage T-Cell Development through Fine-Tuning SCF/Kit Signaling

The phosphatase of regenerating liver (PRL) family of phosphatases, consisting of PRL1, PRL2, and PRL3, represents an intriguing group of proteins being validated as biomarkers and therapeutic targets in human cancer. We have been investigating the role of PRL2 in normal / malignant hematopoiesis and found that PRL2 is important for HSC self-renewal (Kobayashi et al., Stem Cells, 2014). The receptor tyrosine kinase KIT can balance quiescence for HSC maintenance and proliferation for progeny supply. The defects seen in the PRL2-deficient hematopoietic and testis cells recapitulate the phenotype of c-Kit mutant mice, suggesting that the SCF/KIT signaling may be impaired in the absence of PRL2 (Kobayashi et al., Stem Cells, 2014; Dong et al., JBC, 2013). Given that KIT also plays critical role in maintaining postnatal T-lymphopoiesis in thymus, we hypothesized that PRL2 is important for T cell development.Here we report that loss of PRL2 impairs T-lymphopoiesis both in vitro and in vivo. PRL2 deficiency resulted in marked reduction of splenocyte and thymocyte counts compared to wild type (WT) mice. While we observed modest increase in the frequency of early T cell progenitor (ETP), DN2, and DN3 cells in PRL2 deficient thymus, T-cell reconstitution was dramatically decreased after HSC transplantation. T-cell number in the peripheral blood (PB) of recipient mice repopulated with PRL2-null HSCs was 30 times less than that of the WT HSCs (WT: 2288.6±579.8/µl vs PRL2 null: 69.5±22.1/µl, p<0.00001). Although the frequency of donor-derived thymocytes in recipient thymus was 91±6.1% in WT, PRL2 null HSCs contributed only 7.1±4.9% (p<0.00001) in the recipient thymus. By detailed fractionation, surprisingly, chimerism in ETP was comparable between WT and PRL2 null cells (WT: 91.8±10.1% vs PRL2 null: 59.6±13.5%, p<0.01). Importantly, the chimerism of PRL2-null thymocytes fell down to 10% in gated DN2, whereas WT HSCs consistently contributed around 90%, suggesting that the DN1-to-DN2 transition requires PRL2. Next, we evaluated the in vitro T-cell generation by utilizing the Delta-Like1 (DLL1) expressing OP9 (DL-OP9) stromal cells. While wild type KSLs produced massive amount of T-cells (fold increase: 33,000±3371) 22 days following plating onto the DL-OP9, PRL2 null KSLs only generated limited amount of T-cells (fold increase: 1765±665, p<0.0001), demonstrating that PRL2 is important for T-cell proliferation. We also monitored the generation of ETPs from KSLs in DL-OP9 cultures and observed significant expansion of ETPs derived from WT KSLs compared to that of the PRL2 null KSLs (fold increase: 183.8±14.4 vs 12.5±4.3, p<0.001). However, when sorted DN3 cells from WT and PRL2 KO thymus were plated onto DL-OP9, we saw similar increase in cell expansion, suggesting PRL2 regulate early T-cell development.WhilePRL2 is a dual specificity protein phosphatase, its substrates are unknown. To identifyPRL2 substrates in hematopoietic cells, we performed a protein phosphatase substrate trap assay. We utilized a GST-tagged PRL2/CS-DA mutant, in which the catalyticsite cysteine was mutated to serine, so that PRL2 binds to its substrates better, but is unable todephosphorylate them. We found that the mutant PRL2/CS-DA showed enhanced association with KIT than WT PRL2 in Kasumi-1 cells, suggesting that KIT is a potential PRL2 substrate. The PRL2 and KIT interaction was further confirmed by the Immunoprecipitation (IP) assay in 293T cells expressing KIT. We also detected the association of PRL2 with SHP2, CBL and PLC-g in Kasumi-1 cells, which are important regulators of KIT activation and stability. Moreover, PRL2 KO hematopoietic progenitor cells show decreased KIT phosphorylation at tyrosine 703 following SCF stimulation, suggesting that PRL2 may modulate KIT activation in these cells. To evaluate the impact of SCF signal strength on T-cell proliferation, we cultured sorted lympho-primed multipotent progenitor cells (LMPPs) from WT and KO mice onto DLL-Fc coated plates with graded doses of SCF (0.2, 1, 5, 25 ng/ml). The total number of cells generated from SCF treated WT LMPPs was significantly higher than that of the KO LMPPs in a dosage dependent manner. KO exhibited 6 times less sensitive to SCF than WT, indicating that PRL2 fine-tunes SCF signal intensity in early T-cell.Taken together, we have identified a critical role for PRL2 in T-cell proliferation and maintenance through fine-tuning SCF/KIT signaling. DisclosuresNo relevant conflicts of interest to declare.

Activation of the Hypoxia-Inducible Factor Pathway Expands Early Thymic Progenitors

Early thymic progenitors enter the thymus and are exposed to regional hypoxia while they develop in a step-wise manner to mature functional T-cells. Therefore, hypoxia might represent an important component of the highly specialized thymic microenvironment. On the molecular level the hypoxia-inducible factor pathway controls the cellular response to hypoxia. In this pathway, the von-Hippel-Lindau protein (pVHL) continuously mediates the destruction of the transcription factor hypoxia-inducible factor-1α (HIF-1α) under normoxic conditions. Under hypoxia HIF-1α degradation is inhibited leading to the activation of HIF-1α target genes.Others used lck-Cre transgene-mediated conditional in vivo deletion of the Vhl gene to study the role of the oxygen-sensing pathway in developing thymocytes and found normal numbers of early double-negative (DN; CD4-CD8-) thymocytes (Biju et al., Mol Cell Biol, 2004). However, lck-Cre deletion initiates at the DN3 (CD25+CD44-) stage leaving the Vhl locus of very early DN1 (CD25-CD44+), DN2 (CD25+CD44+) and DN3 thymocytes unaltered. Therefore, we here used the ubiquitous hematopoietic deleter strain vav-Cre to investigate the role of pVHL in very early thymocytes (vav-Cre;VhlloxP;loxP mice). Using a PCR-based strategy we confirmed complete deletion of the Vhl gene in this model. We observed unaltered DN1 and DN2 progenitor numbers, however in contrast to the published lck-cre-mediated system we consistently observed an up to twofold expansion of the DN3 cellular compartment. As the hypoxia-inducible factor pathway was shown to modulate NOTCH1 signaling we studied Notch1 expression on Vhl-deficient thymocytes. Strikingly, Notch1 expression was significantly increased on expanded Vhl null DN3 thymocytes. At the DN3 developmental stage selection of cells with an accurately re-arranged T-cell receptor β-locus occurs. Thus, we analyzed pre- and post-β-selection DN3 cells by CD28 staining. Interestingly, we found both pre- and post-β-selection DN3 subpopulations expanded. In order to investigate whether the progenitor expansion is mediated by the lack of HIF-1α inhibition in the Vhl-deficient context we studied DN3 thymocytes in a conditional hematopoietic HIF-1α gain-of-function model (vav-Cre;HIF1dPA). Overexpression of HIF-1α, which is insensitive to pVHL-mediated degradation in vav-Cre;HIF1dPAmice, also resulted in expanded DN3 thymocytes.In summary, we describe novel conditional models to genetically alter the hypoxia-inducible factor pathway within very early thymic progenitors. Genetic Vhl loss led to an expansion of DN3 thymocytes. This DN3 expansion is most likely due to the absence of HIF-1α-inhibition, because HIF-1α overexpression phenocopied the Vhl-deficient DN3 thymocyte expansion. DisclosuresDührsen:Celgene: Honoraria, Research Funding.

Histone Acetylation Mediated by Brd1 Is Crucial for Cd8 Gene Activation during Early Thymocyte Development

During T cell development, Cd8 expression is controlled via dynamic regulation of its cis-regulatory enhancer elements. Insufficient enhancer activity during initial Cd8 activation is known to cause a variegated CD8 expression, thereby generating a CD4+CD8-TCRb-/low population that should appear as CD4+CD8+ double-positive (DP) thymocytes. However, epigenetic and molecular mechanisms involved in initial Cd8 gene activation remain elusive. We previously reported that Brd1, also known as Brpf2, is responsible for the global acetylation of H3K14 as a subunit of the Hbo1 histone acetyltransferase (HAT) complex.In this study, we generated conditional Brd1 knockout mice (Tie2-Cre;Brd1fl/fl mice), in which Brd1 is inactivated in all hematopoietic cells. Although Tie2-Cre;Brd1fl/fl mice were born and grew normally, detailed analysis revealed an abnormal thymocyte differentiation. Deletion of Brd1 resulted in the appearance of CD4+CD8-TCRβ-/low thymocytes that was indistinguishable from DP thymocytes in their properties. Hierarchical clustering of gene expression profile showed that Brd1Δ/Δ CD4+CD8-TCRβ-/low thymocytes retain an expression profile nearly identical to one observed in DP thymocytes. These results indicate that Brd1Δ/Δ CD4+CD8-TCRβ-/low thymocytes correspond to immature thymocytes that would normally appear as CD4+CD8+ DP thymocytes, and was generated because of a variegated activation of Cd8 gene. Importantly, in the gut intraepithelial lymphocyte (IEL) compartment, the population of CD4+CD8aa+ aβT cells, whose generation requires Cd8 gene reactivation in CD4+CD8− helper T cells, were significantly decreased in Tie2-Cre;Brd1fl/fl mice compared to the Tie2-Cre control mice. In addition, the percentage of CD8aa expressing gdT cells was significantly reduced. These findings suggest that Brd1 is also required to initiate Cd8a activation in gdT cells as well as Cd8 reactivation in CD4+T cells.We further conducted retroviral transduction of Brd1 into Brd1Δ/Δ DN3 cells that were flowed by culture on TSt-4/DLL stromal cells. Upon exogenous Brd1 expression, CD8 expression was completely restored and thereby Brd1Δ/Δ DN3 cells differentiated into DP cells.ChIP analysis demonstrated that Brd1 and Hbo1 co-localize at the known enhancers in the Cd8 locus and that their bindings are responsible for acetylation at H3K14. Biochemical results confirmed a presence of Brd1 in Hbo1 HAT complexes. These findings indicate that the Brd1-mediated HAT activity is crucial for efficient activation of Cd8 expression via acetylation at H3K14, which would serve as an epigenetic mark that promotes the recruitment of transcription machineries to the Cd8 enhancers. DisclosuresNo relevant conflicts of interest to declare.

Differential Development of Donor Bone Marrow-Derived Thymocytes after Allogeneic in Utero Hematopoietic Cell Transplantation in the Murine Model

IntroductionIn Utero Hematopoietic Cell Transplantation (IUHCT) is a promising therapeutic strategy for congenital hematopoietic disorders. While mixed allogeneic hematopoietic chimerism with associated donor specific tolerance is routinely achieved by a predominant mechanism of central deletion, the critical events of donor and host thymocyte development have not been determined. In this study, we utilized the murine model of allogeneic IUHCT to analyze donor and host thymocyte development in the context of normal immune ontogeny.MethodsBone marrow (BM) cells (10x106) from C57/BL6 (B6, H2kb) mice were injected intravenously into Balb/c (H2kd) fetuses at embryonic day 14 (E14). E14 B6 fetuses injected with GFP B6 BM were used as congeneic controls. At indicated postnatal ages the thymocytes were delineated by multi-color flow cytometry. Cell apoptosis and proliferation were determined by Annexin V staining and in vivo BrdU incorporation, respectively. T cell alloreactivity was assessed by in vitro and in vivo MLR.ResultsOur findings demonstrate that the thymic processing of donor BM-derived thymocytes differs significantly from host thymocyte processing and from thymocyte development in normal control mice. While the phenotypic development of host thymocytes remained comparable with that of normal control Balb/c mice, the four major subsets of donor thymocytes showed altered distribution, with significantly higher proportions of single positive (SP) cells, and a dramatically lower proportion of CD4+CD8+ double positive (DP) cells, compared to their host-derived counterparts and B6 controls. The extent of the alteration is directly related to both BM chimerism levels and age. Higher levels of chimerism and/or older age are associated with more profound alterations in donor thymocyte distribution. Moreover, DP cells of donor origin show higher apoptosis and lower proliferation than those of the host. Donor TCR gamma/delta cells in DN cells which do not require positive selection based on MHC recognition are relatively increased compared to the host. Moreover, compared with the naive mice and congeneic chimeric mice, the donor BM-derived thymocytes in the allogeneic chimeric mice show increased proportion of DN3 and decreased proportion of DN4, but increased TCRβ+ proportions in both DN3 and DN4 cells, indicating that donor BM-derived thymocyte development is impeded during DN to DP transition, resulting from a MHC-restriction associated mechanism. In addition, in allogeneic chimeric mice, both host and donor BM-derived T cells are tolerant to allogeneic antigens in in vivo and in vitro MLR.ConclusionOur data suggests that in an allogeneic IUHCT system the immune reconstitution of the donor bone marrow-derived thymocytes differs from that of the host cells and that of normal mice. The data supports a mechanism of impaired MHC based positive selection of donor cells by the predominantly host thymic stroma resulting in lack of progression of a higher proportion of donor cells from the DN to DP stage of thymocyte development.Taken together, although donor BM-derived T cells undergo differential thymic development, permanent host-donor two-way tolerance could be achieved in the allogeneic IUHCT mouse model. These findings add to our understanding of the requirements for tolerance induction after IUHCT and have important clinical implications in choosing an optimal donor for IUHCT. DisclosuresNo relevant conflicts of interest to declare.

­­­­­­N-Me, a Long Range T-Cell Specific Oncogenic Enhancer in T-ALL

Over the last years, numerous studies have dissected the mutational landscape of T-cell acute lymphoblastic leukemia (T-ALL) resulting in the identification of numerous oncogenes and tumor suppressors implicated in T-cell transformation. However, most genetic abnormalities found in cancer are located in intergenic regions, whose role in cancer development, if any, remains poorly understood. Here we hypothesized that recurrent cancer-associated intergenic mutations, amplifications and deletions may implicate strong transcriptional regulatory sequences responsible for the activation of key oncogenic factors in the pathogenesis of T-ALL. To address this question, we first used ChIPseq analysis to map the genomic landscape of enhancer sequences controlled by NOTCH1, a critical T-ALL oncogene activated by mutations in over 60% of human T-ALLs. In addition, we performed high resolution aCGH analysis of somatic chromosomal amplifications and deletions in a comprehensive series of 160 T-ALL samples. These analyses revealed recurrent focal duplications at chromosome 8q24 in 8/160 (5%) T-ALL cases in an area devoid of protein-coding genes containing a prominent 1 kb NOTCH1-binding peak. Strikingly, this putative oncogenic element is located +1,427 kb 3’ from the MYC locus and chromatin configuration 3C analysis demonstrated its direct association with the MYC proximal promoter. Multispecies DNA sequence alignment revealed remarkable conservation of this region in mammals, birds and reptiles and local ChIP analysis revealed bona fide active enhancer features including P300 occupancy and high levels of H3K4me1 with low levels of H3K4me3. In addition, detailed analysis of epigenetic marks across 64 hematopoietic and non-hematopoietic cell lines and tissues revealed that his regulatory element is located within a major superenhancer specifically active in T-cells. Based on these results, we proposed that this regulatory sequence, hereby named N-Me for NOTCH-bound MYCenhancer, could function as an important regulatory element driving the activation of MYC downstream of NOTCH1 in T-ALL. Consistently, luciferase reporter assays showed strong, orientation-independent and NOTCH-dependent activation of reporter constructs containing N-Me in association with a -2.5 kb MYC proximal promoter in JURKAT T-ALL cells.Next and to formally test the functional relevance of this enhancer in T-cell development and transformation we generated and characterized N-Me knockout and conditional knockout mice. N-Me null animals were viable and showed a marked and selective reduction in thymus size and cellularity as their only developmental alteration. Detailed immunophenotypic analysis of N-Me knockout thymocytes demonstrated accumulation of double negative 3 (DN3) T cells and marked reductions in double positive and CD4+ and CD8+ single positive cells. Mechanistically, this phenotype was associated with a marked reduction in Myc expression in DN3, DN4 and ISP cells. Moreover, transplantation of N-Me knockout bone marrow hematopoietic progenitors demonstrated that this phenotype is cell autonomous, and can be rescued upon retroviral expression of MYC. Next, we analyzed the role of N-Me in the induction of NOTCH-driven leukemias by transplanting mice with N-Me wild-type and knockout hematopoietic progenitors infected with retroviruses expressing a mutant constitutively active form of NOTCH1 (ΔE-NOTCH1). In this context, mice transplanted with ΔE-NOTCH1-infected N-Me wild type cells developed T-ALL with 100% penetrance 6 weeks after transplant. In contrast, mice transplanted with ΔE-NOTCH1-N-Me knockout cells showed complete resistance to NOTCH1-induced transformation remaining 100% leukemia-free at 15 weeks post-transplant. In addition, secondary deletion of N-Me in established NOTCH1 induced leukemias from tamoxifen-inducible N-Me conditional knockout mice (Rosa26TMCre N-Meflox/flox) induced profound antileukemic effects with extended survival and almost complete suppression of leukemia initiating cell activity. Altogether, these results identify N-Me as the first long range oncogenic enhancer directly implicated in the pathogenesis of human leukemia. DisclosuresNo relevant conflicts of interest to declare.

Acute Myeloid Leukemia Alters Natural Killer Cell Maturation and Functional Activation

Acute myeloid leukemia (AML) is a devastating disease primarily affecting adults. Immune evasion is a major mechanism of AML persistence, and represents a barrier for long-term clinical success. Natural killer (NK) cells are a key component of the innate immune system and hold promise as a tool for effective anti-leukemia therapy. However, to date, the clinical success of NK therapy has been disappointing, indicating additional immune evasion strategies may affect the ability of NK cells to function in patients. We hypothesized that AML may evade the innate immune system by inhibiting NK cell maturation.To evaluate NK function and maturation during AML progression, our lab utilized a novel knock-in model of AML that expresses both Flt3-ITD and Mll-PTD mutations (PTD/ITD), and develops AML with 100% penetrance and recapitulates human disease. For these studies, both primary PTD/ITD mice and transplanted AML blasts were used. For transplant studies, AML or wild-type (WT) control cells (CD45.2+) were transplanted into irradiated C57BL/6 (CD45.1+) naïve recipients. Normal (CD45.1+) NK cells were defined as NK1.1+/CD3- by flow cytometry, and expression of receptors was determined within this population. NK cells in leukemic mice exhibited a reduction in the activating receptors Ly49D and Ly49H in the spleen, blood, LN, and bone marrow. There was also an increase in the inhibitory receptor Ly49C/I and NKG2C/A/E (both p<0.05). However, these alterations were not consistent with all activating and inhibitory receptors, as CD69 was elevated, while NKp46 expression was decreased (both p<0.05). NK function was determined by ELISA measurement of soluble interferon-gamma (IFN-g) production. While there was no change in IFN-g production at baseline, there was surprisingly a significant elevation in IFN-g detected upon stimulation with interleukin-12 and interleukin-18 in the NK cells isolated from leukemic mice as compared to WT control mice (p=0.0004).NK maturation was then evaluated by examining CD11b and CD27 protein expression on NK1.1+/CD3- cells in the spleen, bone marrow, blood, and LN. CD11b+/CD27+ (DP) NK cells were reduced in the spleen as well as bone marrow, blood and LN (p<0.02 for all organs). AML splenocytes had a corresponding increase in NK cells negative for both proteins (DN) when compared to WT control splenocytes (p=0.0004). Previous studies indicate the DP population of NK cells have the highest cytotoxic and cytokine producing functions, while DN NK cells are functionally immature. These alterations were more distinct as AML burden increased in the mice (p=0.0004). Interestingly, there was no significant difference in the most mature CD11b+/CD27- NK population in the spleen, but it there was a significant increase in the CD11b+/CD27- population in the blood (p=0.002). Additional analysis of two transcription factors known to regulate NK development, T-bet and Eomes, was also performed. A reduction in expression of both transcription factors was seen in total NK cells of leukemic mice, as well as NK subsets in the spleen (p<0.02). These data suggest that NK maturation is altered in the presence of AML, and that this alteration becomes more pronounced as leukemic burden increases. Studies evaluating the mechanistic link between AML and NK maturation are ongoing. Future studies will evaluate the efficacy of combinatorial therapies addressing both this imbalance in maturation as well as enhancing recognition to improve NK killing of AML cells. DisclosuresNo relevant conflicts of interest to declare.

The Role of Signal-Transducing Adaptor Protein-2 in Early T Lymphopoiesis in Thymus

Immune cells show quick responses to infection. Many studies concerning cellular and humoral immunity have focused on the regulation of mature lymphocyte function. However, recent studies revealed that the early stage of hematopoiesis plays an important role in the immune system. In bone marrow, hematopoietic stem/ progenitors (HSPC) are targets of pathogen products and danger signals. After the exposure to Gram-negative lipopolysaccharide, the ligand of Toll-like receptor 4, hematopoietic stem cells enter cell-cycle and differentiate into myeloid lineage cells while B lymphopoiesis almost arrests. Little has been known how early T lymphopoiesis in thymus changes. Previously, we cloned signal-transducing adaptor protein-2 (STAP-2) as a c-fms/M-CSFR interacting protein, and found STAP-2 in T lymphocytes or macrophages is crucial for immune responses. The function of STAP-2 is generally recognized under inflammatory condition, interacting with a variety of signaling or transcriptional molecules. We reported that STAP-2 binds to STAT5 for regulation of T cell proliferation, and in macrophages, STAP-2 combines with MyD88 and IκB kinase to activate NF-κB and enhances the production of IL-6 and TNFα. In this study, the effects of STAP-2 on early T progenitors were evaluated using gene-modified mice. All experimental procedures were conducted under specific pathogen-free conditions, according to protocols approved by Institutional Animal Care and Use Committees of Osaka University.We first evaluated the expression level of STAP-2 in murine thymus with quantitative PCR. STAP-2 mRNA was ubiquitously observed all through T cell development, including the CD4- CD8- double-negative (DN) stage. To test the influences on T lymphopoiesis, we generated knock-out and transgenic mice (Tg) that are modified STAP-2 gene expression. In Tg mice, STAP-2 was overexpressed under the control of the Lck proximal promoter. The promoter could drive expression of the inserted cDNA in T lineage cells from the late of DN 2 stage (CD44+ CD25+). We found that thymus was significantly enlarged in Tg mice (46.7 ± 11.15 mg in WT vs 88.1 ± 25.2 mg in Tg), while the number of T lymphocytes in periphery was comparable to wild-type mice (WT). Results from flow cytometric analysis showed STAP-2 enhanced the percentages of DN2 and DN3 (CD44- CD25+) T progenitors, and the actual numbers of DN2, DN3, DN4 (CD44- CD25-) and CD4+ CD8+ double-positive subpopulations. There were no differences between control and knock-out mice in thymus and peripheral bloods. When Lin- Sca1+ cKitHigh HSPC derived from Tg mice were cultured with Delta-like 1-transduced OP9 stromal cells (OP9-DL1) under T cell generation condition, the development of DN4 cells was accelerated (26.9 ± 6.2 % in WT vs 35.2 ± 4.1 % in Tg). Co-cultures from Lin- CD44+ CD25- cKitHigh early T cell progenitors showed the same tendency. These results indicate that STAP-2 regulates the proliferation and differentiation of T progenitors during DN3 to DN4 stage. To elucidate the signaling regulated by STAP-2, microarray experiment with DN3 T progenitors was conducted. The bioinformative approach with Ingenuity Pathway Analysis showed the canonical pathways related with IL-12 signaling, 4-1BB (CD137) signaling and helper T cell differentiation were significantly influenced. Interestingly, we found that STAP-2 affected the distribution of functional T lymphocytes. The ratio of helper CD4+ cells to suppressor CD8+cells in peripheral bloods was lower in Tg mice than that in WT.In summary, we found that STAP-2 regulates the early T lymphopoiesis in thymus. DN2 to DN4 stages of T progenitors increased in STAP-2 transgenic mice, and STAP-2 promoted the differentiation in vitro. Moreover, STAP-2 affected the cell decision in development to helper CD4+ cells or suppressor CD8+ cells. Our study indicates the up-regulation of STAP-2 under inflammatory condition might be crucial for immune response at the early stage of T lymphopoiesis. Further study would clarify the precise molecular mechanisms of the enhancement of T lymphopoiesis by STAP-2. DisclosuresNo relevant conflicts of interest to declare.

Canine CD4+CD8+ double-positive T cells can develop from CD4+ and CD8+ T cells

For a long time the expression of the CD4 and CD8 receptor on peripheral blood T cells was thought to be mutually exclusive. However, in canine peripheral blood, similar to other species as swine or human for example, mature CD4+CD8+ double-positive (dp) T cells exist which simultaneously express both surface receptors and have features of activated T cells. Canine CD4+CD8+dp T cells are heterogeneous and can be divided into three subpopulations by their intensity of CD4 and CD8α expression: CD4brightCD8αbright, CD4dimCD8αbright and CD4dimCD8αdim. The number of CD4+CD8α+dp T cells increases after in vitro stimulation of canine peripheral blood mononuclear cells (PBMC) raising the question of their progenitor(s). Thus, the aim of our study was to characterize the progenitor(s) of canine CD4+CD8α+dp T cells. By cell tracing experiments we identified both CD4+ single-positive (sp) and also CD8α+sp T cells as progenitors of canine CD4+CD8α+dp T cells after in vitro stimulation. CD4+sp T cells almost exclusively upregulate a CD8αα homodimer, whereas CD8α+sp T cells can become CD4+CD8αβ+ or CD4+CD8αα+. Even in the absence of other cells, highly purified CD4+sp T cells can become double-positive upon in vitro stimulation, whereas highly purified CD8α+sp T cells fail to do so. However, CD8α+sp T cells can additionally express CD4 when stimulated in the presence of CD4−CD8α− double-negative (dn) cells or more efficiently when stimulated in the presence of CD4+sp T cells. Soluble factors secreted by CD4+sp T cells are sufficient for the upregulation of CD4 on CD8α+sp T cells, but direct cell–cell contact between CD4+sp and CD8α+sp T cells is more efficient. mRNA analysis shows that additional CD4 expression on CD8α+sp T cells results from de novo synthesis. Thus, uptake of soluble CD4 or trogocytosis is less likely as mechanism for generation of canine double-positive T cells. CD4+CD8α+dp T cells are highly activated independent of their origin except when generated in coculture of CD8α+sp T cells with CD4−CD8α−dn cells. Overall, in dog, CD4+sp T cells are the more potent progenitors of CD4+CD8α+dp T cells compared to CD8α+sp T cells.

GATA-3 dose-dependent checkpoints in early T cell commitment.

GATA-3 expression is crucial for T cell development and peaks during commitment to the T cell lineage, midway through the CD4(-)CD8(-) (double-negative [DN]) stages 1-3. We used RNA interference and conditional deletion to reduce GATA-3 protein acutely at specific points during T cell differentiation in vitro. Even moderate GATA-3 reduction killed DN1 cells, delayed progression to the DN2 stage, skewed DN2 gene regulation, and blocked appearance of the DN3 phenotype. Although a Bcl-2 transgene rescued DN1 survival and improved DN2 cell generation, it did not restore DN3 differentiation. Gene expression analyses (quantitative PCR, RNA sequencing) showed that GATA-3-deficient DN2 cells quickly upregulated genes, including Spi1 (PU.1) and Bcl11a, and downregulated genes, including Cpa3, Ets1, Zfpm1, Bcl11b, Il9r, and Il17rb with gene-specific kinetics and dose dependencies. These targets could mediate two distinct roles played by GATA-3 in lineage commitment, as revealed by removing wild-type or GATA-3-deficient early T lineage cells from environmental Notch signals. GATA-3 worked as a potent repressor of B cell potential even at low expression levels, so that only full deletion of GATA-3 enabled pro-T cells to reveal B cell potential. The ability of GATA-3 to block B cell development did not require T lineage commitment factor Bcl11b. In prethymic multipotent precursors, however, titration of GATA-3 activity using tamoxifen-inducible GATA-3 showed that GATA-3 inhibits B and myeloid developmental alternatives at different threshold doses. Furthermore, differential impacts of a GATA-3 obligate repressor construct imply that B and myeloid development are inhibited through distinct transcriptional mechanisms. Thus, the pattern of GATA-3 expression sequentially produces B lineage exclusion, T lineage progression, and myeloid-lineage exclusion for commitment.

Defects in pro-apoptotic genes, Bim or Bax/Bak, cause accumulation of TCRβhi DN4 cells in thymus (HEM4P.241)

Abstract Regulated induction of apoptosis during thymocyte development is critical to prevent the generation of self-reactive T cells whilst maintaining a repertoire of T cells that can promote protective immunity. Although the pro-apoptotic Bcl-2 family member Bim has been shown to promote apoptotic negative selection of self-reactive thymocytes, the role of Bim in this process is controversial. Here, we have investigated the role of Bim and downstream mediators Bax and Bak on thymocyte selection. Surprisingly, the absence of Bim results in a striking accumulation of DN4 cells with surface TCRβhi feature in the thymus. Similar results were observed in mice with a T cell specific loss of Bax in a Bak knock out background. DN4 cells from Bim-/- or LckCreBaxfl/flBak-/- mice cultured with OP9-DL1 cells largely failed to generate DP cells relative to WT DN4 cells. Further, using a dLckCre system, in which Cre is expressed late in the DP stage, revealed that conditional knock out of Bim or Bax did not result in an accumulation of DN4-TCRβhi cells. Combined, these data suggest that accumulation of DN4-TCRβhi cells in either Bim-/- or LckCreBaxfl/flBak-/- are the result of cells having failed negative selection that downregulate CD4 and CD8 coreceptors. Such a strategy would inherently limit their TCR avidity and reduce their potential for autoreactivity. Our current studies are examining the persistence and location of DN-TCRβhi cells in the periphery of Bim-/- or LckCreBaxfl/flBak-/- mice.

A role for interleukin-15Rα signaling in the homeostasis of early T cell progenitors (LYM7P.716)

Abstract A deficiency in interleukin-15 receptor α (IL-15Rα) expression is associated with an overall decrease in thymic cellularity. To further investigate this defect, we examined T cell progenitors in the thymus of wild-type (WT) and IL-15Rα KO mice. We identified a significant reduction specifically within the subset of CD27high cells in the CD44highCD25- double negative (DN1) population of T cell progenitors in the absence of IL-15Rα. Since T cell development begins early in ontogeny, we also examined the thymus of WT and IL-15Rα KO neonates (&amp;lt;24hrs post-birth). Surprisingly, we found no differences in the total numbers of neothymocytes or CD27high DN1 cells suggesting that the aforementioned deficiencies are acquired over time. Finally, while IL-15Rα functions largely through the transpresentation of IL-15, it also has the capacity to signal directly. To determine which of these two roles might be involved in the long-term homeostasis of thymic progenitors, we used a transgenic mouse strain that expresses a chimeric receptor composed of the extracellular domain of IL-15Rα and the intracellular domain of the IL-2Rα, thus efficiently disrupting IL-15Rα signaling while maintaining transpresentation. We found that, similar to IL-15Rα KO mice, IL-15Rα signaling transgenics also exhibited a significant decrease in the subset of CD27high DN1 cells suggesting that direct signaling through IL-15Rα is required for optimal homeostasis of early T cell progenitors in the thymus.

CCR6 supports migration and differentiation of a subset of DN1 early thymocyte progenitors but is not required for thymic nTreg development

T-cell selection and development occurs as precursor cells journey through the thymus and interact with stromal cells residing in distinct microenvironments. Although the chemokines CCL19, CCL21, CCL25 and CXCL12 are known to have major roles in intrathymic migration of thymocytes and thymocyte precursors, the significance of other chemokines such as CCL20, which is also expressed in the thymus, is unknown. This is of particular interest given that the thymus is the location of development of the natural regulatory T-cell (nTreg) population and that the CCL20 receptor CCR6 has an important role in peripheral tolerance via control of Treg cell migration. However, whether the CCL20/CCR6 axis has a role in the formation or migration of nTregs in the thymus is unknown. In this study, we addressed this by analyzing expression of CCR6/CCL20 within the thymus and assessing their role in thymocyte development using Ccr6(-/-) mice. CCL20 is predominately expressed in the thymic medulla and CCR6 expression is restricted to nTregs and a subset of early thymocyte progenitor double-negative 1 (DN1) cells (CD4(-)CD8(-)CD25(-)CD44(+)CD117(+)). Ex vivo chemotaxis assays indicated that these two subsets were apparently the sole subsets of thymocytes responsive to CCL20. The data indicate that nTreg frequencies and localization are unperturbed by deletion of Ccr6. However, in Ccr6(-/-) thymi, reduced frequencies of DN2 and DN3 cells, the thymocyte progenitor subsets that follow the DN1 stage, were apparent. Together, these data indicate that CCR6 has a supplementary role in coordination of early thymocyte precursor migration events important for normal subsequent thymocyte precursor development, but is not required for normal nTreg development.

IFN-γ–Producing and IL-17–Producing γδ T Cells Differentiate at Distinct Developmental Stages in Murine Fetal Thymus

γδ T cells develop at the double-negative (DN) 2 and DN3 stages and acquire functions to produce IL-17 and IFN-γ in fetal thymus. However, the relationship between differentiation stages and their functions was unclear. In this study, we found that, although IFN-γ-producing and IL-17-producing γδ T cells developed from DN2 cells, only IFN-γ-producing γδ T cells developed from DN3 cells, indicating the direct generation of IL-17-producing γδ T cells from the DN2 stage, not through the DN3 stage. Single-cell analysis revealed that DN2 cells contained heterogeneous γδ T cell precursors with or without an ability to develop IL-17 producers. Inactivation of B cell leukemia/lymphoma 11b, a zinc finger transcription factor responsible for transition from early to late stages of DN2 cells, completely abrogated the development of IL-17-producing γδ T cells, although a unique subset of IFN-γ-producing γδ T cells expressing a high level of promyelocytic leukemia zinc finger was able to develop. Thus, our results reveal that γδ T cells are functionally differentiated to IFN-γ and IL-17 producers at different developmental stages in fetal thymus.

Characterization Of RING Finger E3-Ubiquitin Ligase Cereblon In Hematopoietic Regulation

Characterization of RING finger E3-Ubiquitin Ligase Cereblon in Hematopoietic RegulationCereblon is a RING-domain E3 ubiquitin ligase (UbL) that is the direct protein target for thalidomide and lenalidomide. Inhibition of this molecule mediates antiproliferative activity in myeloma cells and in activated B-cell-like subtype diffuse large B-cell lymphoma. In addition to its anticancer properties, lenalidomide potentiates T-cell effector function through the engagement of the CD28-mediated co-stimulatory pathway by a CRBL-dependent mechanism that is poorly characterized. Human and animal studies show CRBL to be ubiquitously expressed in the hematopoietic compartment and in neurons, where a nonsense mutation causes a mild autosomal recessive non-syndromic intellectual disability. Ubiquitin ligase activity underlies suppression of growth factor signaling and maintains homeostasis within the hematopoietic compartment. C-Cbl, cbl-b, GRAIL, and ITCH represent four partially overlapping E3-UbL regulators of cytokine signaling and hematopoietic regulation. To understand the role of CRBL in hematopoetic development and function, we studied a CRBL-deficient mouse strain. All mice used in this study were bred and maintained under specific pathogen-free conditions according to institutional guidelines. The strategy used to create germline crbn deletion (crbl-/-) was described previously, although these mice were not investigated for aberrations in hematopoietic development. First, crbl-/- mice were viable, fertile and normal in appearance without limb malformations. Consistent with a role for CRBL in the hematopoietic development, crbl-/- mice exhibited marked changes in their hematopoietic profiles, including lymphoid hyperplasia, a markedly expanded peripheral white blood cell count and neutrophil expansion. In the T-cell compartment, splenic lymphocytes numbers were increased with demargination of the T-cells into B-cell zones by immunohistochemical staining. Double-negative thymocytes ratio were altered with an accumulation of DN1 and DN3 cells and lower DN4. In contrast, differentiated cell lineages such as mature T-cells, including CD4 and CD8 single-positive (SP) thymocytes, SP peripheral T-cells and naïve and memory T-cells were similar in number to wild-type littermates. The mature T-cells, similar to Cbl-b KO mice showed superior proliferation and IL-2 production in the absence of CD28 co-ligation. Collectively, these histological changes are indicative of a role for crbl in negatively regulating cytokine and receptor signaling events that control cell proliferation or survival. Interestingly, this phenotype overlaps partially with c-cbl and cbl-b KO mice suggesting that they could be coordinately regulating similar pathways. In summary, our findings demonstrate a novel role for crbl, the molecular target of thalidomide and lenalidomide in hematopoiesis and suggest that this molecule may act by an unknown mechanism in concert with other E3 UbLs involved in cytokine receptor signaling. This study provides the first characterization of crbl genetic deficiency in a murine model and demonstrates a unique regulatory relationship between CRBL and other known E3 UbLs. Disclosures:McDaniel:Celgene: Employment.